Recombinant protein containing a C-terminal fragment of Plasmodium MSP-1

ABSTRACT

The invention relates to a recombinant protein fabricated in a baculovirus system, of which the essential constitutive polypeptide sequence is that of a C-terminal fragment of 19 kilodalton (p19) of the surface protein 1 (protein MSP-1) of the merozoite parasite of the  Plasmodium  type, particularly  Plasmodium falciparum , which is infectious for humans, said C-terminal fragment remaining normally anchored at the surface of the parasite at the end of its penetration phase into human erythrocytes, in the occurrence of an infectious cycle. Said recombinant protein is applicable to the production of vaccines against malaria.

The invention relates to novel active principles for vaccines derivedfrom the major surface protein in merozoite forms of a Plasmodium whichis infectious for mammals, especially humans, more generally termedMSP-1.

MSP-1 has already been the subject of a number of studies. It issynthesised in the schizont stage of Plasmodium type parasites, inparticular Plasmodium falciparum, and is expressed in the form of one ofthe major surface constituents of merozoites both in the hepatic stageand in the erythrocytic stage of malaria (1, 2, 3, 4). Because of theprotein's predominant character and conservation in all known Plasmodiumspecies, it has been suggested that it could be a candidate forconstituting anti-malarial vaccines (5, 6).

The same is true for fragments of that protein, particularly the naturalcleavage products which are observed to form, for example duringinvasion by the parasite into erythrocytes of the infected host. Amongsuch cleavage products are the C-terminal fragment with a molecularweight of 42 kDa (7, 8) which is itself cleaved once more into anN-terminal fragment with a conventional apparent molecular weight of 33kDa and into a C-terminal fragment with a conventional apparentmolecular weight of 19 kDa (9) which remains normally fixed to theparasite membrane after the modifications carried out on it, viaglycosylphosphatidylinositol (GPI) groups (10, 11).

It is also found at the early ring stage of the intraerythrocyticdevelopment cycle (15, 16), whereby the observation was made that the 19kDa fragment could play a role which is not yet known, but which isdoubtless essential in re-invasive processes. This formed the basis forhypotheses formed in the past that that protein could constitute aparticularly effective target for possible vaccines.

It should be understood that the references frequently made below to thep42 and p19 proteins from a certain type of Plasmodium are understood torefer to the corresponding C-terminal cleavage products of the MSP-1protein of that Plasmodium or, by extension, to products containingsubstantially the same amino acid sequences, obtained by geneticrecombination or by chemical synthesis using conventional techniques,for example using the “Applied System” synthesiser, or by “Merrifield”type solid phase synthesis. For convenience, references to “recombinantp42” and “recombinant p19” refer to “p42” and “p19” obtained bytechniques comprising at least one genetic engineering step.

Faced with the difficulty of obtaining large quantities of parasites forP. falciparum and the impossibility of cultivating P. vivax in vitro, ithas become clear that the only means of producing an anti-malariavaccine is to resort to techniques which use recombinant proteins orpeptides. However, MSP-1 is very difficult to produce whole because ofit large size of about 200 kDa, a fact which has led researchers tostudy the C-terminal portion, the (still unknown) function of which isprobably the more important.

Recombinant proteins concerning the C-terminal portion of the P.falciparum MSP-1 which have been produced and tested in the monkey (12,40, 41) are: a p19 fused with a glutathione-S-transferase produced in E.coli (40);

-   -   a p40 fused with a glutathione-S-transferase produced in E. coli        (12);    -   a p19 fused with a polypeptide from a tetanic anatoxin and        carrying auxiliary T cell epitopes produced in S. cerevisiae        (12);    -   a p42 produce in a baculovirus system (41).

A composition containing a p19 protein fused with aglutathione-S-transferase produced in E. coli combined with alum orliposomes did not exhibit a protective effect in any of six vaccinatedAotus nancymai monkeys (40).

A composition containing a p42 protein fused with aglutathione-S-transferase produced in E. coli combined with a Freundcomplete adjuvant did not exhibit a protective effect in two types ofAotus monkeys (A. nancymai and A. vociferans) when administered to them.The p19 protein produced in S. cerevisiae exhibited a protective effectin two A. nancymai type Aotus monkeys (12). In contrast, there was noprotective effect in two A. vociferans type Aotus monkeys.

Some researchers (Chang et al.) have also reported immunisation testscarried out in the rabbit using a recombinant p42 protein produced in abaculovirus system and containing one amino acid sequence in common withP. falciparum (18). Thus these latter authors indicate that in therabbit that recombinant p42 behaves substantially in the same way as theentire recombinant MSP-1 protein (gp195). This p42 protein incombination with a Freund complete adjuvant has been the subject matterof a vaccination test in a non-human primate susceptible to infection byP. falciparum, Aotus, lemurinus grisemembra (40). The results showedthat 2 of 3 animals were completely protected and the third, whileexhibiting a parasitemia which resembled that of the controls, had alonger latent period. It is nevertheless risky to conclude to aprotective nature in man of the antibodies thus induced against theparasites themselves. It should be remembered that there are currentlyno very satisfactory experimental models in the primate for P. vivax andP. falciparum. The Saimiri model, developed for P. falciparum and P.vivax, and the Aotus model for P. falciparum, are artificial systemsrequiring the parasite strains to be adapted and often requiringsplenectomy of the animals to obtain significant parasitemia. As aresult, the vaccination results from such models can only have a limitedpredictive value for man.

In any event, what the real vaccination rate would be which couldpossibly be obtained with such recombinant proteins is alsoquestionable, bearing in mind the discovery—reported below—of thepresence in p42s from Plasmodiums of the same species, and moreparticularly in the corresponding p33s, of hypervariable regions whichwould in many cases render uncertain the immunoprotective efficacy ofantibodies induced in individuals vaccinated with a p42 from aPlasmodium strain against an infection by other strains of the samespecies (13).

It can even be assumed that the high polymorphism of the N-terminalportion of p42 plays a significant role in immune escape, often observedfor that type of parasite.

The aim of the present invention is to produce vaccinating recombinantproteins which can escape these difficulties, the protective effect ofwhich is verifiable in genuinely significant experimental models or evendirectly in man.

More particularly, the invention provides vaccinating compositionsagainst a Plasmodium type parasite which is infectious for man,containing as an active principle a recombinant protein which may or maynot be glycosylated, whose essential constituent polypeptide sequenceis:

-   -   either that of a 19 kilodalton (p19) C-terminal fragment of the        surface protein 1 of the merozoite form (MSP-1 protein) of a        Plasmodium type parasite which is infectious for man, said        C-terminal fragment remaining normally anchored to the parasite        surface at the end of its penetration phase into human        erythrocytes in the event of an infectious cycle;    -   or that of a portion of that fragment which is also capable of        inducing an immune response which can inhibit in vivo        parasitemia due to the corresponding parasite;    -   or that of an immunologically equivalent peptide of said p19        fragment or said portion of that fragment; and        said recombinant protein further comprises conformational        epitopes which are unstable in a reducing medium and which        preferably constitute the majority of the epitopes recognised by        human antiserums formed against the corresponding Plasmodium.

The presence of such conformational epitopes could play an importantrole in the protective efficacy of the active principle of the vaccines.They are particularly found in the active principles which exhibit theother characteristics defined above, when they are produced in abaculovirus system. If needs be, it is mentioned below that theexpression “baculovirus vector system” means the ensemble constituted bythe baculovirus type vector itself and the cell lines, in particularcells of insects transfectable by a baculovirus modified by a sequenceto be transferred to these cell lines resulting in expression of thattransferred sequence. Preferred examples of these two partners in thebaculovirus system have been described in the article by Longacre et al.(19). The same system was used in the examples below. It goes withoutsaying, of course, that variations in the baculovirus and in the cellswhich can be infected by the baculovirus can be used in place of thoseselected.

In particular, the recombinant protein is recognised by human antiserumsformed against the corresponding Plasmodium or against a homologousPlasmodium when it is in its non reduced state or in a reduced nonirreversible state, but is not recognised or is only recognised to aslight extent by these same antiserums when it is irreversibly reduced.

The unstable character of these conformational epitopes in a reducingmedium can be demonstrated by the test described below in the examples,in particular in the presence of β-mercaptoethanol. Similarly, theexamples below describe the experimental conditions applicable to obtainirreversible reduction of the proteins of the invention.

From this viewpoint, the recombinant protein produced by Longacre et al.(14) can be used in such compositions. It should be remembered that S.Longacre et al. succeeded in producing a recombinant p19 from the MSP-1of P. vivax in a baculovirus vector system containing a nucleotidesequence coding for the p19 of Plasmodium vivax, in particular bytransfecting cultures of insect cells [Spodoptera frugiperda (Sf9) line]with baculovirus vectors containing, under the control of the polyhedrinpromoter, a sequence coding for the peptide sequences defined below,with the sequences being placed in the following order in thebaculovirus vector used:

-   -   a 35 base pair 5′ terminal fragment of the polyhedrin signal        sequence, in which the methionine codon for initiating        expression of this protein had been mutated (to ATT);    -   a 5′-terminal nucleotide fragment coding for a 32 amino acid        peptide corresponding to the N-terminal portion of MSP-1,        including the MSP-1 signal peptide;    -   either a nucleotide sequence coding for p19, or a sequence        coding for the p42 of the MSP-1 protein of Plasmodium vivax,        depending on the case, these sequences also being provided with        (“anchored” forms) or deprived of (soluble forms) 3′ end regions        of these nucleotide sequences, whose end C-terminal expression        products are reputed to play an essential role in anchoring the        final p19 protein to the parasite membrane;    -   2 TAA stop codons.

For p42, the sequences derived from the C-terminal region of MSP-1extend consequently from amino acid Asp 1325 to amino acid Leu 1726(anchored form) or to amino acid Ser 1705 (soluble form) and for p19,the sequences extend from amino acid Ile 1602 to amino acid Leu 1726(anchored form) or to amino acid Ser 1705 (soluble form) it beingunderstood that the complete amino acid sequences of p42 and p19, whoseinitial and terminal amino acids have been indicated above follow fromthe gene of the Belem isolate of P. vivax which has been sequenced (20).

Similar results were obtained using, in the same vector systems,nucleotide sequences coding for the p42 and p19 of Plasmodium cynomolgi.The interest in P. cynomolgi is twofold: it is a parasitic species veryclose to P. vivax which is infectious for the macaque. It can alsoinfect man. Further, access to the natural hosts of P. cynomolgi, rhesusmonkeys and toque macaques, is also possible, to test the efficacy ofthe protection of MSP-1 from P. cynomolgi in natural systems. The rhesusmonkey is considered to be one of the most representative species forimmune reactions in man.

In particular, excellent results have been obtained in vaccination testscarried out using the toque macaque with two recombinant polypeptides:soluble p42 and, in particular, soluble p19 derived from P. cynomolgi,respectively produced in a baculovirus system and purified on anaffinity column with monoclonal antibodies recognising the correspondingregions of the native MSP-1 protein. The following observations weremade: six monkeys immunised with only p19 (three monkeys) and the p19and p42 together (three monkeys) all exhibited practically sterileimmunity after challenge infection. The results obtained in the threemonkeys immunised with p42 were less significant. Two of them were asabove, but since the third exhibited a lower parasitemia than thecontrols immunised with a PBS buffer in the presence of Freund adjuvant(3 monkeys) or not immunised (3 monkeys), it was less clear.

A second challenge infection showed that the monkeys which had receivedp19 alone were protected for at least six months. A second vaccinationtest with p19 in combination with alum in this system (toque macaque P.cynomolgi) exhibited significant protection for 2 of the 3 monkeys. Thisis the first time that MSP-1 or another recombinant antigen hasdemonstrated a protective effect in the presence of alum (42).

The particularly effective test results carried out with the macaquewith recombinant polypeptides produced in a baculovirus system using arecombinant p19 from P. cynomolgi showed that recombinant polypeptidesrespectively containing recombinant p19s from other Plasmodiums mustbehave in the same manner. They are more meaningful for malaria in manthan the results from tests carried out with P. vivax or P. falciparumin their “artificial hosts”.

Baculovirus recombinant proteins derived from a C-terminal MSP-1 portion(p19) have a very significant antimalarial protective effect in anatural system, which constitutes the most representative model forevaluating the protective effect of MSP-1 for man.

The protective effect obtained can be further improved if the p19 formis deprived of the hypervariable region of the N-terminal portion ofp42, the effect of which can be deleterious in natural situations inwhich the vaccinated subject is confronted by a great deal ofpolymorphism. Further, p19 appears to possess specific epitopes whichare not present in p42.

The 19 kDa C-terminal fragment, the sequence of which is present in theactive principle of the vaccine, can be limited to the sequence for thep19 itself, in the absence of any polypeptide sequence normally upstreamof the p19 sequence in the corresponding MSP-1 protein. Clearly, though,the essential constituent polypeptide sequence of the active principlecan also comprise a polypeptide sequence for the C-terminal sidebelonging to the 33 kDa (p33) N-terminal fragment still associated withthe p19 in the corresponding p42, before natural cleavage of the latter,if the presence of this fragment does not modify the immunologicalproperties of the active principle of the vaccine. As will be seenbelow, in particular in the description of the examples, the C-terminalsequences of the p33 in various strains of the same species ofPlasmodium (see the C-terminal portion of the peptide sequences of“region III” in FIG. 4) also have a degree of homology or substantialconservation of the sequence, for example of the order of at least 80%,in different varieties of Plasmodiums which are infectious for man, suchthat they do not fundamentally modify the vaccinating properties of theactive principle (the sequence ofo which corresponds to region IV inFIG. 4), in particular using the hypothesis which follows from thisfigure; that the presumed cleavage site between the p19 and region IIIof the p33 is located between the leucine and asparagine residues in aparticularly well conserved region (LNVQTQ).

Normally the C-terminal polypeptide sequence of the p33, when it ispresent, comprises less than 50 amino acid residues, or even less than35, preferably less than 10 amino acid residues.

In contrast, the essential constituent polypeptide sequence of theactive principle of the vaccine need not comprise all of the sequencecoding for p19, naturally providing that the latter retains the abilityto induce antibodies which protect against the parasite. In particular,the molecular weight of the “fragment portion” is 10 to 25 kDa, inparticular 10 to 15 kDa. Preferably, this polypeptide fragment portioncontains at least one of the two EGF (Epidermal Growth Factor) regions.

Clearly, the skilled person could distinguish between active fragmentsand those which would no longer be so, in particular experimentally byproducing modified vectors containing inserts with different lengthsoriginating from the p19, respectively isolated from the fragmentsobtained from the sequence coding for p19, by reaction with appropriaterestriction enzymes, or by exonucleolytic enzymes which would be kept incontact with the fragment coding for p19 for differing periods; thecapacity of the expression products from these inserts in thecorresponding eukaryotic cells, in particular in insect cells,transformed by the corresponding modified vectors, to exert a protectiveeffect can then be tested, in particular under the experimentalconditions which are described below in the examples. In particular, theexpression products of these inserts must be able to inhibit aparasitemia induced in vivo by the corresponding whole parasite.

Thus, the invention includes all vaccinating compositions in which theessential constituent polypeptide sequence of the active principle isconstituted by a peptide which can induce a cellular and/or humoral typeimmunological response equivalent to that produced by p19 or a fragmentas defined above, provided that the addition, deletion or substitutionin the sequence of certain amino acids by others would not cause a largemodification of the capacity of the modified peptide—hereinafter termedthe “immunologically equivalent peptide”—to inhibit said parasitemia.

The p19 fragment can naturally also be associated at the N-terminal sideor the C-terminal side or via a peptide bond to a further plasmoidalprotein fragment having a vaccinating potential (such as Duffy bindingprotein from P. vivax (29) or EBA-175 from P. falciparum (30) and (31),one region of which is specifically rich in cysteine), provided that itscapacity to inhibit parasitemia normally introduced in vivo by thecorresponding parasite is not altered but is amplified.

Upstream of the N-terminal end of p19, the fragment coding for p19 or aportion thereof can also contain a peptide sequence which is differentagain, for example a C-terminal fragment of the signal peptide used,such as that for the MSP-1 protein. This sequence preferably comprisesless than 50 amino acids, for example 10 to 40 amino acids.

These observations pertain in similar fashion to the p19s from otherPlasmodium, in particular P. falciparum, the dominant species of theparasites, responsible for one of the most serious forms of malaria.

However, the techniques summarised above for producing a recombinant p19from P. vivax or P. cynomolgi in a baculovirus system are difficult totranspose unchanged to producing a recombinant p19 of P. falciparum in asatisfactory yield, if only to obtain appreciable quantities which willallow immunoprotective tests to be carried out.

The invention also provides a process which overcomes this problem to alarge extent. It also becomes possible to obtain much higher yields ofP. falciparum p19—and other Plasmodiums where similar difficulties areencountered—using a synthetic nucleotide sequence substituting thenatural nucleotide sequence coding for the p19 of Plasmodium falciparumin an expression vector of a baculovirus system, this syntheticnucleotide sequence coding for the same p19, but being characterized bya higher proportion of G and C nucleotides than in the naturalnucleotide sequence.

In other words, the invention follows from the discovery that expressionof a nucleotide sequence coding for a p19 in a baculovirus system isapparently linked to an improved compatibility of successive codons inthe nucleotide sequence to express with the “cellular machinery” of thehost cells transformable by the baculovirus, in the manner of thatobserved for the natural nucleotide sequences normally contained inthese baculovirus and expressed in the infected host cells; hence thepoor expression, or even total absence of expression of a native P.falciparum nucleotide sequence; hence also a possible explanation of themore effective expression observed by Longacre et al. (14) for the p19of P. vivax in a baculovirus system and, as the inventors have alsoshown, of the P. cynomolgi sequence from corresponding native p19nucleotide sequences, because of their relatively much higher amounts ofG and C nucleotides than those of the native nucleotide sequences codingfor the p19 of P. falciparum.

The invention thus more generally provides a recombinant baculovirustype modified vector containing, under the control of a promotercontained in said vector and able to be recognised by cellstransfectable by said vector, a first nucleotide sequence coding for asignal peptide exploitable by a baculovirus system, characterized by asecond nucleotide sequence downstream of the first, also under thecontrol of the promoter and coding for the peptide sequence:

-   -   either of a 19 kilodalton (p19) C-terminal fragment of the        surface protein 1 of the merozoite form (MSP-1 protein) of a        Plasmodium type parasite other than Plasmodium vivax which is        infectious for man, said C-terminal fragment remaining normally        anchored to the parasite surface at the end of its penetration        phase into human erythrocytes in the event of an infectious        cycle;    -   or of a portion of that peptide fragment provided that the        expression product from the second sequence in a baculovirus        system is also capable of inducing an immune response which can        inhibit in vivo parasitemia due to the corresponding parasite;    -   or of an immunologically equivalent peptide of said C-terminal        peptide fragment (p19) or said peptide fragment portion by        addition, deletion or substitution of amino acids not resulting        in a large modification of the capacity of said immunologically        equivalent peptide to induce a cellular and/or humoral type        immunological response similar to that produced by said p19        peptide fragment or said portion of said fragment; and        said nucleotide sequence having, if necessary, a G and C        nucleotide content in the range 40% to 60%, preferably at least        50%, of the totality of the nucleotides from which it is        constituted. This sequence can be obtained by constructing a        synthetic gene in which the natural codons have been changed for        codons which are rich in G/C without modifying their translation        (maintaining the peptide sequence).

The nucleotide sequence, provided by a synthetic DNA, may have at least10% of modified codons with respect to the natural gene sequence or cDNAwhile retaining the characteristics of the natural translated sequence,i.e., maintaining the amino acid sequence.

It is not excluded that this G and C nucleotide content could be furtherincreased provided that the modifications resulting therefrom as to theamino acid sequence of the recombinant peptide—or immunologicallyequivalent peptide—produced do not result in a loss of immunologicalproperties, or protective properties, of the recombinant proteinsformed, in particular in the tests which will be described below.

These observations naturally apply to other Plasmodium which areinfectious for man, in particular those where the native nucleotidesequences coding for corresponding p19s would have T and A nucleotidecontents which are poorly compatible with effective expression in abaculovirus system.

The sequence coding for the signal used can be that normally associatedwith the native sequence of the Plasmodium concerned. But it can alsooriginate from another Plasmodium, for example P. vivax or P. cynomolgior another organism if it can be recognised as a signal in a baculovirussystem.

The sequence coding for p19 or a fragment thereof in the vector underconsideration is, in one case, deprived of the anchoring sequence of thenative protein to the parasite from which it originates, in which casethe expressed protein is generally excreted into the culture medium(soluble form). It is also remarkable in this respect that under theconditions of the invention, the soluble and anchored forms of therecombinant proteins produced, in particular when they are from P.falciparum or P. cynomolgi or P. vivax, tend to form oligomers, thisproperty possibly being at the origin of the increased immunogenicity ofthe recombinant proteins formed.

The invention also concerns vectors in which the coding sequencecontains the terminal 3′end sequence coding for the hydrophobicC′-terminal end sequence of the p19 which is normally implicated in theinduction of anchoring the native protein to the cell membrane of thehost in which it is expressed. This 3′-terminal end region can also beheterologous as regards the sequence coding for the soluble p19 portion,for example corresponding to the 3′-terminal sequence from P. vivax orfrom another organism when it codes for a sequence which anchors thewhole of the recombinant protein produced to the cell membrane of thehost of the baculovirus system used. An example of such anchoringsequences is the GPI of the CD59 antigen which can be expressed in thecells of Spodoptera frugiperda (32) type insects or the GPI of a CD14human protein (33).

The invention also, naturally, concerns recombinant proteins, theseproteins comprising conformational epitopes recognised by human serumsformed against the corresponding Plasmodium.

In general, the invention also concerns any recombinant protein of thetype indicated above, provided that it comprises conformational epitopessuch as those produced in the baculovirus system, in particular thosewhich are unstable in a reducing medium.

The invention also, naturally, concerns said recombinant proteins,whether they are in their soluble form or in the form provided with ananchoring region, in particular to cellular hosts used in thebaculovirus system.

The invention also encompasses oligomers spontaneously produced in thebaculovirus systems used or produced a posteriori, using conventionalprotein oligomerisation techniques. The most commonly used techniqueinvolves glutaraldehyde. However, any conventional system for bridgingbetween the respective amine and carboxyl functions in proteins can beused. As an example, any of the techniques described in European patentapplication EP-A-0 602 079 can be used.

The term “oligomer” means a molecule containing 2 to 50 monomer units,each of the monomer units containing p19 or a fragment thereof, asdefined above, capable of forming an aggregate. The invention alsoencompasses any conjugation product between a p19 or a p19 fragment asdefined above, and a carrier molecule—for example apolylysine-alanine—for use in producing vaccines, via bonds which arecovalent or otherwise. The vaccinating compositions using them also formpart of the invention.

The invention still further concerns vaccine compositions using theseoligomeric or conjugated recombinant proteins, including proteins fromPlasmodium vivax, these observations also extending to oligomers ofthese recombinant proteins.

The invention also encompasses compositions in which the recombinantproteins defined above are associated with an adjuvant, for example analum. Recombinant proteins containing the C-terminal end region allowingthem to anchor to the membrane of the cells in which they are producedare advantageously used in combination with lipids which can formliposomes appropriate to the production of vaccines. Without beinglimiting, lipids described, for example, in the publication entitled“Les liposomes aspects technologique, biologique et pharmacologique”[Liposomes: technological, biological and pharmacological aspects] by J.Delattre et al., INSERM, 1993, can be used.

The presence of the anchoring region in the recombinant protein, whetherit is a homologous or heterologous anchoring region as regards thevaccinating portion proper, encourages the production of cytophilicantibodies, in particular IgG_(2a) and IgG_(2b) type in the mouse whichcould have a particularly high protective activity, so that associatingthe active principles of the vaccines so constituted with adjuvantsother than the lipids used to constitute the liposome forms could bedispensed with. This amounts to a major advantage, since liposomes canbe lyophilised under conditions which enable them to be stored andtransported, without the need for chains of cold storage means.

Other characteristics of the invention will become clear from thefollowing description of examples of recombinant proteins of theinvention and the conditions under which they can be produced. Theseexamples are not intended to limit the scope of the invention.

Description of the Construction of PfMSP1_(p19)S (Soluble) (Soluble p19from P. falciparum)

The recombinant construction PfMSP1_(p19)S contains the DNAcorresponding to 8 base pairs of the leader sequence and the first 32amino acids of the MSP-1 of Plasmodium vivax from Met, to Asp₃₂ (Belemisolate; Del Portillo et al., 1991, P. N. A. S., 88, 4030) followed byGluPhe due to the EcoR1 site connecting the two fragments. This isfollowed by the synthetic gene described in FIG. 1, coding thePlasmodium falciparum MSP1_(p19) from Asn₁₆₁₃ to Ser₁₇₀₅ (Uganda-PaloAlto isolate; Chang et al., 1988, Exp. Parasitol., 67, 1). Theconstruction is terminated by two TAA stop codons. This constructiongave rise to a recombinant protein which was secreted in the culturesupernatant from infected cells.

In the same manner and for comparison, a recombinant construction wasproduced under conditions which were similar to those used to producethe p19 above, but working with a coding sequence consisting of a directcopy of the corresponding DNA of the P. falciparum strain (FUP)described by Chang et al., Exp. Parasit. 67, 1; 1989. The natural genecopy (from asparagine 1613 to serine 1705) was formed from the nativegene by PCR.

FIG. 1A shows the sequences of both the synthetic gene (Bac19) and the“native gene” (PF19).

It can be seen that 57 codons of the 93 codons of the native sequencecoding for the p19 from P. falciparum were modified (the thirdnucleotide in 55 of them and the first and third nucleotides in theother 2 codons). New codons were added to the 5′ end to introduce thepeptide signal under the conditions indicated above and to introduce anEcoRI site for cloning, and similarly two stop codons were added whichwere not present in the P. falciparum p19 to obtain expressiontermination signals. The individual letters placed above successivecodons correspond to the respective successive amino acids. Asterisks(*) show the stop codons. Vertical lines indicate the nucleotides whichare the same in the two sequences

Description of the PfMSP1_(p19)A Construction (Anchored GPI) (Anchoredp19 of P. falciparum)

The PfMSP1_(p19)A construction had the characteristics of that aboveexcept that the synthetic sequence (FIG. 1B) codes for the MSP1_(p19) ofPlasmodium falciparum (Uganda-Palo Alto isolate) from Asn₁₆₁₃ to Ile₁₇₂₆followed by two TAA stop codons. This construction gave rise to arecombinant protein which was anchored in the plasma membrane ofinfected cells by a glycosyl phosphatidyl inositol (GPI) type structure.

FIG. 1C represents the PfMSP1_(p19)S recombinant protein sequence beforecutting out the signal sequence.

FIG. 1D represents the PfMSP1_(p19)S recombinant protein sequence aftercutting out the signal sequence.

The amino acids underlined in FIGS. 1C and 1D originate from the EcoR1site used to join the nucleotide sequences derived from the N-terminalportion of the MSP-1 of P. vivax (with signal sequence) and theMSP-1_(p19) of P. falciparum.

FIG. 2—The soluble recombinant PfMSP1_(p19) antigen purified byimmunoaffinity was analysed by immunoblot using SDS-PAGE in the presence(reduced) or absence (non reduced) of β-mercaptoethanol. Samples werecharged onto gel after heating to 95° C. in the presence of 2% SDS.Under these conditions only covalent type bonds (disulphide bridges) canresist disaggregation. The left hand blot was revealed with a monoclonalantibody which reacted with a linear epitope of natural p19. The righthand blot was revealed with a mixture of 13 human antisera originatingfrom subjects with acquired immunity to malaria due to Plasmodiumfalciparum. These results show that the recombinant baculovirus moleculecan reproduce conformational epitopes in the form of a polymer themajority of which are recognised by human antiserum.

FIG. 2B: Immunoblot Analysis with Human Antiserum of RecombinantPurified MSP-1 p19 from P. vivax and P. cynomolgi Under Non Reduced(NR), Reduced Only in the Charging Medium (R) and Irreversibly Reduced(IR) Conditions

This work was based on the idea that the baculovirus expression systemcorrectly reproduced the conformational epitopes present in vivo on theC-terminal portion of MSP-1 in large amounts. The best means ofmeasuring this property (which may be the only possible means in theabsence of native purified proteins corresponding to p19) was to studythe reactivity of the recombinant proteins with the antiserum ofindividuals exposed to malaria, this reflecting the native proteins as“seen” by the human immune system.

Thus soluble recombinant PvMSP-1 p19 and PcMSP-1 p19 antigens purifiedby immunoaffinity were analysed by immunoblot using SDS-PAGE (15%) inthe presence (reduced) or absence (non reduced) of DTT. Samples werecharged onto gel after heating to 95° C. in the presence of 2% SDS. Theirreversible reduction was carried but as follows: the protein wasresuspended in 0.2 M Tris-HCl, pH 8.4, 100 mM DTT, 1.0% SDS and heatedfor 30 minutes at 70° C. After diluting with water, acrylamide was addedto a final concentration of 2 M and the mixture was incubated undernitrogen in the dark for 1 hour at 37° C. The immunoblot was revealedwith a mixture of 25 human antisera originating from subjects with anacquired immunity to malaria due to Plasmodium vivax. V and Crespectively designate proteins derived from the MSP-1 of P. vivax andP. cynomolgi. It should be noted that irreversibly reduced recombinantproteins exhibited no reactivity with human antiserum while nonirreversibly reduced proteins or non reduced proteins exhibited goodreactivity. (The non reduced Pv MSP-1 p19 was a little weak since in itsglycosylated state it does not bind well to nitro-cellulose paper).These results show that recognition of baculovirus MSP-1 p19 moleculesby human antiserum is largely if not completely dependent onconformational epitopes sensitive to reduction which are reproduced inthis system.

FIG. 3—The soluble PvMSP1_(P42) recombinant antigen (Longacre et al.,1994, op. Cit.) was incubated for 5 hours at 37° C. in the presence ofprotein fractions derived from merozoites of P. falciparum and separatedby isolectrofocussing. The samples were then analysed by immunoblot inthe presence (reduced) or absence (non reduced) of β-mercaptoethanol.Isolectrofocussing fractions 5 to 12, and two total merozoite extractsmade in the presence (Tex) or absence (T) of detergent, were analysed.The immunoblot was revealed with monoclonal antibodies specific forMSP1_(P42) and _(p19) of P. vivax. The results suggest that there is aproteolytic activity in the P. falciparum merozoites which can beextracted with detergent. Digestion of p42 in certain fractions appearto cause polymerisation of the digestion products (p19); thispolymerisation is probably linked to the formation of disulphide bridgessince in the presence of β-mercaptoethanol, the high molecular weightforms disappear in favour of a molecule of about 19 kDa (Tex-R). The p19polymerisation observed in these experiments could thus be an intrinsicproperty of this molecule in vivo.

FIG. 3B: The Differential Contribution of p42 and p19 Antigens to the P.vivax Anti-MSP-1 Human Response

Recognition of P. vivax MSP-1 p42 and p19 antigens by the antiserum ofindividuals with an acquired immunity to P. vivax was compared using theELISA inhibition technique as follows: a mixture of 25 human antiseraoriginating from subjects with an acquired immunity to malaria due to P.vivax was diluted to 1:5000 and incubated for 4 hours at ambienttemperature either alone, or in the presence of a 1 mM purified P. vivaxrecombinant p42 or p19. This mixture was transferred to a microtitrewell which had been coated for 18 hours at 4° C. with 500 ng·ml⁻¹ ofpurified absorbed recombinant p42 or p19, and incubated for 30 minutesat ambient temperature. After washing with PBS containing 0.1% of Tween20, a goat anti-mouse IgG conjugated with peroxidase was added and themixture was incubated for 1 hour at 37° C. The enzymatic activity wasrevealed by reading the optical density at 492 nm. The percentageinhibition was calculated based on values of 100% of antiserum activitywith the coated antiserum on the microtitre plate in the absence of acompeting antigen. Statistical data were calculated using a Statviewprogram. Each bar represents the average percentage inhibition of a pairof competing/absorbed antigens based on 4 to 12 determinations; thevertical lines correspond to a 95% confidence interval. Asterisks (*)designate the antigens produced in the presence of tunicamycin, thuswith no N-glycosylation. The important parameters of these measurementswere the dilution of the antiserum by 1:5000 which is in the regionwhich is sensitive to ELISA curves and the competing antigenconcentrations of 1 mM which includes competition by low affinityepitopes. Thus these data reflect the maximum resemblance between thetwo compared antigens. The results show that the majority, if not all ofthe p42 epitopes recognised by the human antiserum are present on thep19 since in the presence of the latter, the reactivity of the humanantiserum against p42 is inhibited as much as by the p42 antigen itself.In contrast, however, about 20% of the p19 epitopes recognised by humanantiserum were not or were not accessible on the p42, since thereactivity of the human antiserum against the p19 was much lessinhibited by p42 than by p19 itself. Such specific epitopes of p19 canbe constituted or revealed only after cleaving the p42 into p19 and p33.These results were not affected by glycosylation showing that the effectis really due to a difference between the peptide components of p19 andp42 and not to a difference in glycosylation. These results underlinethe fact that p19 has a distinct immunological identity to p42.

Description of the PcMSP1_(p19)S (Soluble) Construction (Soluble p19 ofP. cynomolgi)

The DNA used for the above construction was obtained from a clone of thePlasmodium cynomolgi ceylonesis strain (22-23). This strain had beenmaintained by successive passages through its natural host (Macacasinica) and cyclic transmissions via mosquitoes (27).

Blood parasites in the mature schizont stage were obtained from infectedmonkeys when the parasitemia had attained a level of 5%. They were thenpurified using the methods described in (25). The DNA was then extractedas described in (26).

A 1200 base pair fragment was produced using a PCR reaction using theoligonucleotides underlined in FIG. 4 originating from P. vivax. The 5′oligonucleotide comprised an EcoRI restriction site and the 3′oligonucleotide comprised two synthetic TAA stop codons followed by aBglII restriction site. This fragment was introduced by ligation and viathese EcoRI and BglII sites into the pVLSV₂₀₀ plasmid already containingthe signal sequence for the MSP-1 protein of P. vivax (19). The newplasmid (pVLSV₂₀₀C₄₂) was used to analyse the DNA sequences.

The P. cynomolgi and the corresponding P. vivax sequences were aligned.The black arrows designate the presumed primary and secondary cleavagesites. They were determined by analogy with known sites in P. falciparum(27, 28). The vertical lines and horizontal arrows localise the limitsof the four regions which were studied. Region 4 corresponded to thesequence coding for the P. cynomolgi p19. Glycosylation sites are boxedand the preserved cysteines are underlined. The lower portion of FIG. 4shows the percentage identity between the two isolates of P. vivax andP. cynomolgi.

The recombinant construction PcMSP1_(p19)S contains the DNAcorresponding to 8 base pairs of the leader sequence and the first 32amino acids of the MSP-1 of Plasmodium vivax from Met₁ to Asp₃₂ (Belemisolate; Del Portillo et al., 1991, P. N. A. S., 88, 4030) followed byGluPhe, due to the EcoR1 site, connecting the two fragments. This isfollowed by the sequence coding for the Plasmodium cynomolgi MSP1_(p19)from Lys₂₇₆ to Ser₃₈₀ (Ceylon strain). The construction was terminatedby two TAA stop codons. This construction gave rise to a recombinantprotein which was secreted in the culture supernatant of infected cells.

Purification of Recombinant PfMSP1_(p19) Protein by ImmunoaffinityChromatography with a Monoclonal Antibody Specifically Recognising thep19 of Plasmodium falciparum

The chromatographic resin was prepared by binding 70 mg of a monoclonalantibody (obtained from a G17.12 hybridoma deposited at the CNCM[National Collection of Microorganism Cultures] (Paris, France) on the14 Feb. 1997, registration number 1-1846; this G17.12 hybridoma wasconstructed from X63 Ag8 653 myeloma producing IgG 2a/k recognising theP. falciparum p19) to 3 g of activated CNBr-Sepharose 4B (Pharmacia)using standard methods detailed in the procedure employed by Pharmacia.The culture supernatants containing the soluble PfMSP1p19 were batchincubated with the chromatographic resin for 16 hours at 4° C. Thecolumn was washed once with 20 volumes of 0.05% NP40, 0.5 M of NaCl,PBS; once with 5 volumes of PBS and once with 2 volumes of 10 mM ofsodium phosphate, pH 6.8. Elution was carried out with 30 ml of 0.2 Mglycine, pH 2.2. The eluate was neutralised with 1 M sodium phosphate,pH 7.7 then concentrated by ultrafiltration and dialysed against PBS. Topurify the anchored PfMSP1p19, all of the washing and elution solutionscontained a supplemental 0.1% of 3-(dimethyl-dodecylammonio)-propanesulphonate (Fluka).

Recombinant Plasmodium vivax (p42 and p19) MSP1 Vaccination Test in theSquirrel Monkey Saimiri sciureus

This vaccination test was carried out on male non splenectomised 2 to 3year old Saimiri sciureus boliviensis monkeys. Three monkeys wereinjected 3 times intramuscularly at 3 week intervals with a mixture ofabout 50 to 100 μg each of recombinant soluble PvMSP1_(p42) and _(p19)(19), purified by immunoaffinity. Complete and incomplete Freundadjuvant was used as follows: 1^(st) injection: 1:1 FCA/FIA; 2^(nd)injection: 1:4 FCA/FIA; 3^(rd) injection: FIA. These adjuvantcompositions were then mixed 1:1 with the antigen in PBS. Five controlmonkeys received the glutathione-S-transferase (GST) antigen produced inE. coli using the same protocol. The challenge infection was carried outby injecting 2×10⁶ red blood cells infected with an adapted Plasmodiumvivax strain (Belem) 2.5 weeks after the final injection. The protectionwas evaluated by determining parasitemia daily in all animals byexamining smears stained with Giemsa.

The curves in FIG. 5 show the variation in the measured parasitemia asthe number of parasited red blood cells per microlitre of blood (up theordinate, logarithmic scale) as a function of the time passed afterinfection (in days). Curve A corresponds to the average values observedin the three vaccinated monkeys; curve B corresponds to the averagevalues in the five control monkeys.

An examination of the Figure shows that the effect of the vaccinationwas to greatly reduce the parasitmisa.

Recombinant Plasmodium cynomolgi (p42 and p19) MSP1 Vaccination Test inthe Toque Macaque Macaca sinica

Fifteen captured monkeys were used as follows: (1) 3 animals injectedwith 100 μg of soluble PcMSP1_(p42); (2) 3 animals injected with 35 μg(1^(st) injection) or 50 μg (2^(nd) and 3^(rd) injections) of solublePcMSP1_(p42); (3) 3 animals injected with a mixture of PcMSP1_(p42) andp19; (4) 3 animals injected with adjuvant plus PBS; (5) 3 animals notinjected. Complete and incomplete Freund adjuvant was used in theprotocol described above. Injections were intramuscular at 4 weekintervals. The challenge infection was made by injecting 2×10⁵ red bloodcells infected with Plasmodium cynomolgi 4 weeks after the lastinjection. Protection was evaluated by determining parasitemia daily inall animals by examining the parasitemia with Giemsa. Parasitemia wereclassified as negative only after counting 400 smear fields. Theparasitemia were expressed as a percentage of parasitised red bloodcells.

FIGS. 6A-6G show the results obtained. Each of them shows parasitemia(expressed as the percentage of parasitised red blood cells along theordinate on a logarithmic scale) observed in the challenge animals as afunction of the time after infection (in days along the abscissa).

The results relate to:

-   -   in FIG. 6A; non vaccinated control animals;    -   FIG. 6B relates to animals which received a saline solution also        containing Freund adjuvant;    -   FIG. 6C is a superposition of FIGS. 6A and 6B, with the aim of        highlighting the relative results resulting from administration        of Freund adjuvant to the animals (the variations are clearly        not significant);    -   FIG. 6D provides the results obtained at the end of vaccination        with p42;    -   FIG. 6E concerns animals vaccinated with p19 alone;    -   finally, FIG. 6F concerns animals vaccinated with a mixture of        p19 and p42.

The p42 certainly induced a certain level of protection. However, asshown in FIGS. 6E and 6F, the protection conferred by the recombinantp19 of the invention was considerably better.

The hypothesis can be formulated that the improved protection resultingfrom secondary cleavage of p42 which is accompanied by revealing freecysteine which, as a result, forms intermolecular bridges giving rise top19 multimers which are highly characteristic of this form inrecombinant proteins of the three species tested.

The numbers used to produce graphs (6A-6F) are given in FIG. 6G.

P. cynomolgi Toque Macaque Vaccination Test; Second Challenge Infectionof Monkeys Vaccinated with p19 Alone and Controls (FIG. 8)

Six months later, with no other vaccination, the 3 macaques whichreceived the p19 MSP-1 alone with FCA/FIA (FIG. 6E) and the 3 macaqueswhich received a saline solution containing Freund adjuvant (FIG. 6B)and 2 new unaffected unvaccinated monkeys underwent a new challengeinfection by injecting 1×10⁶ red blood cells infected with Plasmodiumcynomolgi. Protection was evaluated by determining parasitemia daily inall animals by examining Giemsa smears. The parasitemia were classifiednegative only after counting 400 smear fields. The parasitemia wereexpressed as the percentage of parasitised red blood cells (the figuresused to produce graphs 8A-C are given in FIG. 8D). The six immunisedanimals which underwent challenge infection six months earlier had nodetectable parasitemia except for 1 animal in each group which exhibiteda parasitemia of 0.008% for 1 day (FIGS. 8A and 8B). The two unaffectedcontrols exhibited a conventional parasitemia with a maximum of 0.8% andfor 21 days (FIG. 8C). Thus the 3 animals vaccinated with the MSP-1 p19were also protected six months later than the 3 controls which exhibiteda complete conventional infection after the first challenge infection,despite the absence of or a very slight parasitemia after the firstchallenge infection. These results suggest that the protection periodfor p19 is at least six months.

Vaccination Test with p19 in Association with Alum in the P. cynomolgiToque Macaque System (FIG. 9)

The previous positive protection results were obtained using complete(FCA) or incomplete (FIA) Freund adjuvant. However, the only adjuvantwhich is currently allowed in man is alum. For this reason, we carriedout a vaccination test with P. cynomolgi MSP-1 p19 in the toque macaquein the presence of alum as the adjuvant. Six captured macaques were usedas follows: (1) 3 animals injected with 4 doses of 50 mg of recombinantP. cynomolgi MSP-1 p19 with 20 mg of alum; (2) 3 animals injected 4times with physiological water and 10 mg of alum. The injections wereintramuscular at 4 week intervals. The challenge infection was made byinjecting 2×10⁵ red blood cells infected with P. cynomolgi 4 weeks afterthe last injection. Protection was evaluated by daily determination ofparasitemia in all animals by examining Giemsa smears. The parasitemiawere classified negative only after counting 400 smear fields.Parasitemia were expressed as the percentage of parasitised red bloodcells. The results of this experiment were as follows. 2 of the 3macaques immunised with recombinant p19 with alum had about 30 timesless total parasitemia during the infection period (FIGS. 9A and 9B)than the 3 control macaques immunised with physiological water and alum(FIG. 9D) after the challenge infection. The third macaque immunisedwith p19 (FIG. 9C) was not very different from the controls. For thevaccination test using Plasmodium cynomolgi p19 in the toque macaque,macaca sinica, described in FIG. 9, the data used to produce the graphs(9A-9D) are given in (FIG. 9E). While the results are a little lessspectacular than the preceding results (FIGS. 6, 8), this is the firsttime that significant protection has been observed for recombinant MSP-1with alum.

FIG. 10: Vaccination Test with a Recombinant Plasmodium falciparum p19in the Squirrel Monkey

Twenty Saimiri sciureus guyanensis (squirrel monkeys) of about 3 yearsold raised in captivity were used as follows: (1) 4 animals injectedwith 50 mg of soluble Pf MSP-1 p19 in the presence of Freund adjuvant asfollows: 1^(st) injection: 1:1 FCA/FIA; 2^(nd) injection: 1:4 FCA/FIA;3^(rd) injection: FIA. These adjuvant compositions were then mixed with1:1 antigen in PBS; (2) 2 control animals received Freund adjuvant asdescribed for (1) with only PBS; (3) 4 animals injected with 50 mg ofsoluble Pf MSP-1 p19 in the presence of 10 mg of alum (Alu-Gel-S,Serva); (4) 2 control animals received 10 mg of alum with only PBS; (5)4 animals injected with about 50-100 mg of GPI anchored Pf MSP-1 p19reconstituted into liposomes as follows: 300 mmoles of cholesterol and300 mmoles of phosphatidyl choline were vacuum dried and resuspended in330 mM of N-octylglucoside in PBS with 1.4 mg of Pf MSP-1 p19, GPI. Thissolution had been dialysed against PBS with adsorbent Bio-Beads SM-2(Bio-Rad) and the liposomes formed were concentrated by centrifuging andresuspended in PBS The 1^(st) injection was made with fresh liposomeskept at 4° C. and the 2^(nd) and 3^(rd) injections were made withliposomes which had been frozen for preservation; (6) 2 animals injectedwith control liposomes made in the same way, in the absence of the p19,GPI antigen as described for (5); (7) 2 animals injected withphysiological water. Three intramuscular injections were made at 4 weekintervals. The challenge infection was made by injecting 1×10⁶ red bloodcells infected with Plasmodium falciparum. Protection was evaluated bydetermining parasitemia daily in all animals by examining the Giemsasmears. Parasitemia were expressed as the percentage of parasitised redblood cells. The results of this vaccination test are shown in FIGS. 10,A-G.

The groups immunised with p19 in Freund adjuvant or liposomedemonstrated similar parasitemia to the control groups after a challengeinfection (one animal (number 29) vaccinated with p19 in Freund adjuvantdied several days after challenge infection for reasons independent ofvaccination (cardiac arrest”. Irregularities in administration of theantigen in these 2 groups (poor Freund emulsion, congealed liposomes)did not allow the significance of these results to be completelyevaluated. In the alum group, 2 animals showed total parasitemia for theduration of the infection about 4 times less than the controls, 1 animalabout 3 times less and 1 animal was similar to the controls. Thisexperiment was a little difficult to interpret due to the variability inthe controls, probably due to the strain of parasite used for thechallenge infection which would not have been quite adapted to the nonsplenectomised Saimiri model developed only recently in Cayenne.However, the real effect with alum, although imperfect, is encouragingin that our antigens seem to be the only recombinant P. falciparum MSP-1versions which currently have shown a certain effectiveness incombination with alum.

Vaccination Test with a Recombinant Plasmodium falciparum p19 in theSquirrel Monkey (Same Test as for FIG. 10)

Monkeys bred in captivity were injected intramuscularly with 1 ml ofinoculum twice at 4 week intervals as follows: (1) 4 animals injectedwith 50 μg of soluble PfMSP1p19 in the presence of Freund adjuvant asfollows: 1^(st) injection: 1:1 FCA/FIA; 2^(nd) injection: 1:4 FCA/FIA;and mixed then 1:1 with the antigen in PBS; (2) 4 animals injected with50 μg of soluble PfMSPp19 in the presence of 10 mg of alum; (3) 4animals injected with about 50 μg of GPI anchored PfMSP1p19reconstituted into liposomes composed of 1:1 molar cholesterol andphosphatidyl choline. The animals were bled 17 days after the secondinjection.

Red cells from a squirrel monkey with 30% parasitemia due to P.falciparum (with the mature forms in the majority) were washed with PBSand the residue was diluted 8 times in the presence of 2% SDS and 2%dithiothreitol and heated to 95° before being charged onto apolyacrylamide gel of 7.5% (separation gel) and 4% (stacking gel). Aftertransfer to nitrocellulose, immunoblot analysis was carried out withantisera as follows: (1) pool of antisera of 4 monkeys vaccinated withsoluble PfMSP1p19 in Freund adjuvant, twentieth dilution; (2) pool ofantisera of 4 monkeys vaccinated with soluble PfMSP1p19 in alumadjuvant, twentieth dilution; (3) pool of antisera of 4 monkeysvaccinated with anchored PfMSP1p19 in liposomes, twentieth dilution; (4)monoclonal antibody, which reacts with a linear epitope of PfMSP1p19, 50mg/ml; (5) SHI90 antisera pool originating from about twenty monkeysrepeatedly infected with P. falciparum and which had become unaffectableby any subsequent infection with P. falciparum, five hundredth dilution;(6) antiserum pool of unaffected monkeys (never exposed to P.falciparum), twentieth dilution.

The results show that the 3 antiserum pools of monkeys vaccinated withPfMSP1p19 reacted strongly and specifically with very high molecularweight complexes (diffuse in the stacking gel) and present in parasiteextracts containing more mature forms. These results support thehypothesis that a specific aggregate of PfMSP1p19 is present in vivocomprising epitopes which are reproduced in recombinant PfMSP1p19molecules synthesised in the baculovirus system, in particularoligomeric forms thereof.

FIG. 7 also illustrated these results. It shows immunoblots produced ongel. The first three gel tracks illustrate the in vivo response ofmonkeys to injections of p19 [(1) with Freund adjuvant, (2) with alum,(3) in the form of a liposome] and in particular the existence of highmolecular weight complexes supporting the hypothesis of in vivoaggregation of p19 in the form of an oligomer, specific to thematuration stage (when p42 is cut into p19 and p33).

This vaccination test also comprises a third injection identical to theprevious injections. The injection with Freund adjuvant contained onlyFIA.

There were two animal controls for each group, namely: 2 control animalsinjected with PBS and Freund adjuvant; 2 control animals injected withPBS and alum; 2 control animals injected with liposomes without protein;and two control animals injected with PBS without adjuvant. Protectionwas evaluated as described above.

FIG. 7B: The data for this Figure were derived from the squirrel monkeyP. falciparum I vaccination test (FIG. 10 below). The numbers correspondto the individual monkeys noted in FIG. 10. The techniques and methodsfor this Figure were the same as for FIG. 7 except that the individualantiserum for each monkey was tested after three injections the day ofthe proof injection and the SHI antiserum was diluted by 1:250. Theresults show that the antiserum for 4 monkeys vaccinated with p19 andalum reacted strongly and specifically with very high molecular weightcomplexes while the monkeys of other groups vaccinated with p19 andFreund adjuvant or liposomes showed only a little reactivity with thesecomplexes. Since the monkeys vaccinated with p19 and alum were also thebest protected, this reactivity with the high molecular weight complexesappeared to indicate a protective effect, despite one monkey in thegroup not being protected with respect to the controls and that anotherwas only partially protected.

The invention also, of course, concerns other applications, for examplethose described below with respect to certain of the examples, althoughthese are not limiting in character.

Therapy

The recombinant molecule PfMSP1p19 can be used to produce specificantibodies which can possibly be used by passive transfer fortherapeutics for severe malaria due to P. falciparum when there is arisk of death.

Diagnostics

The recombinant molecules PvMSP1p42, PvMSP1p19 and PfMSPp19 derived frombaculovirus can and have been used to produce specific murine monoclonalantibodies. These antibodies, in combination with polyclonalanti-MSP1p19 antisera originating from another species such as therabbit or goat can form the basis of a semi-quantitative diagnostic testfor malaria which can distinguish between malaria due to P. falciparum,which can be fatal, and malaria due to P. vivax, which is generally notfatal. The principle of this test is to trap and quantify any MSP-1molecule containing the p19 portion in the blood.

In this context, the advantages of the MSP1p19 molecule are as follows:

-   (i) it is both extremely well conserved in the same species and    sufficiently divergent between different species to enable specific    species reactants to be produced. No cross reaction has been    observed between antibodies derived from PfMSP1p19 and PvMSP1p19;-   (ii) the function of MSP1p19, while not known with precision, seems    to be sufficiently important that this molecule does not vary    significantly or is deleted without lethal effect for the parasite;-   (iii) it is a major antigen found in all merozoites and thus it must    in principle be detectable even at low parasitemia and    proportionally to the parasitemia;-   (iv)since the recombinant MSP1p19 molecules derived from baculovirus    appear to reproduce more of the native structure of MSP1P19, the    antibodies produced against these proteins will be well adapted to    diagnostic use.

The microorganisms identified below have been deposited under Rule 6.1of the Treaty of Budapest of 1 Feb. 1996, under the followingregistration numbers: Identification reference Registration numbersPvMSP1p19A I-1659 PvMSP1p19S I-1660 PfMSP1p19A I-1661 PfMSP1p19S I-1662PcMSP1p19S I-1663

The invention also concerns the use of these antibodies, preferablyfixed to a solid support (for example for affinity chromatography) forthe purification of type p19 peptides initially contained in a mixture.

Purification means bringing this mixture into contact with an antibody,dissociating the antigen-antibody complex and recovering the purifiedp19 type peptide.

The invention also concerns vaccine compositions, also comprisingmixtures of proteins or fragment, in particular mixtures of the type:

-   -   P. falciparum p19 and P. vivax p19;    -   P. falciparum p19 and P. falciparum p42, the latter if necessary        being deprived of its most hypervariable regions;    -   P. vivax p19 and P. vivax p42, the latter if necessary being        deprived of its most hypervariable regions;    -   P. falciparum p19 and P. falciparum p42, the latter if necessary        being deprived of its most hypervariable regions, and P. vivax        p19 and P. vivax p42, the latter if necessary being deprived of        its most hypervariable regions.

In the present case, the most hypervariable regions are defined asregions I or region II and all or part of region III, the portion ofregion III which is preferably deleted being that which is juxtaposed toregion II (the conserved portion being located to the side of theC-terminal of p33, close to the p19). Regions II and III are illustratedin FIG. 4.

The invention is not limited to the production of human vaccine. It isalso applicable to the production of veterinary vaccine compositionsusing the corresponding proteins or antigens derived from parasiteswhich are infectious for mammals and products under the same conditions.It is known that infections of the same type, babesiosis, also appear incattle, dogs and horses. One of the antigens of the Babesia species hasa high conformational homology (in particular in the two EFG-like andcysteine-rich domains) and functional homology with a protein portion ofMSP-1 [(36), (37) and (38)].

Examples of veterinary vaccines using a soluble antigen against suchparasites have been described (39).

It goes without saying that the p19s used in these mixtures can also bemodified as described in the foregoing when considered in isolation.

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The invention also concerns hybridomas secreting specific antibodiesselectively recognising the p19 of a MSP-1 protein in the merozoite formof a Plasmodium type parasite which is infectious for man other thanPlasmodium vivax and which does not recognise Plasmodium vivax.

In particular, these hybridomas secrete monoclonal antibodies which donot recognise Plasmodium vivax and which specifically recognisePlasmodium falciparum p19.

The invention also concerns a hybridoma characterized in that itproduces a specific antibody which specifically recognises the p19 of P.vivax and the p19 of P. cynomolgi. A F10-3 hybridoma has beenconstructed from the X63 Ag8 653 myeloma producing IgG 2b/k recognisingthe p42 glycoprotein of Plasmodium vivax.

1-45. (canceled)
 46. A baculovirus vector comprising: (a) a promoter;(b) a synthetic polynucleotide comprising a synthetic sequence encodinga 19 kilodalton C-terminal fragment of a Plasmodium falciparum merozoitsurface protein 1 (MSP-1) and having a GC content of 40% to 60%; and (c)a polynucleotide encoding a signal sequence of a Plasmodium MSP-1protein.
 47. The baculovirus vector of claim 46, wherein the syntheticsequence has a GC content of 50%.
 48. The baculovirus vector of claim46, wherein the synthetic sequence has at least 10% of modified codonswith respect to a natural gene sequence or a cDNA of the 19 kilodaltonC-terminal fragment of a Plasmodium falciparum merozoite surface protein1 (MSP-1) while retaining a natural amino acid sequence thereof.
 49. Thebaculovirus vector of claim 46, wherein the polynucleotide encoding saidsignal sequence is a polynucleotide encoding a Plasmodium falciparumMSP-1 protein.
 50. A baculovirus vector, comprising: (a) a promoter; (b)a synthetic polynucleotide consisting essentially of SEQ ID NO:1encoding a 19 kilodalton C-terminal fragment of Plasmodium falciparummerozoite surface protein 1 (MSP-1) and having a GC content of 40% to60%; and (c) a polynucleotide encoding a signal sequence of a PlasmodiumMSP-1 protein.
 51. The baculovirus vector of claim 50, wherein thesynthetic polynucleotide has a GC content of 50%.
 52. The baculovirusvector of claim 50, wherein the polynucleotide encoding said signalsequence is a polynucleotide encoding a Plasmodium falciparum MSP-1protein.
 53. A baculovirus vector, comprising: (a) a promoter; (b) asynthetic polynucleotide consisting essentially of SEQ ID NO:1 encodinga 19 kilodalton C-terminal fragment of Plasmodium falciparum merozoitesurface protein 1 (MSP-1) and having a GC content of 40% to 60%; (c) apolynucleotide sequence coding for at most 50 C-terminal amino acids ofa 33 kilodalton N-terminal fragment of a Plasmodium falciparum merozoitesurface protein 1 (MSP-1) positioned upstream of the syntheticpolynucleotide; and (d) a polynucleotide encoding a signal sequence of aPlasmodium MSP-1 protein.
 54. The baculovirus vector of claim 53,wherein the polynucleotide coding for said C-terminal amino acids has atmost 35 amino acids.
 55. The baculovirus vector of claim 53, wherein thepolynucleotide coding for said C-terminal amino acids has at most 10amino acids.
 56. The baculovirus vector of claim 53, wherein thepolynucleotide encoding the signal sequence is a polynucleotide encodinga Plasmodium falciparum MSP-1 protein.
 57. A synthetic polynucleotidecomprising a synthetic sequence encoding a 19 kilodalton C-terminalfragment of a Plasmodium falciparum merozoite surface protein 1 (MSP-1)having a total GC content of 40% to 60% and a polynucleotide encoding asignal sequence of a Plasmodium MSP-1 protein.
 58. The syntheticpolynucleotide of claim 57, wherein the synthetic polynucleotidesequence has a GC content of 50%.
 59. The synthetic polynucleotide ofclaim 57, wherein the polynucleotide encoding the signal sequence is apolynucleotide encoding a Plasmodium falciparum MSP-1 protein.
 60. Thesynthetic polynucleotide of claim 57, wherein the synthetic sequence hasat least 10% of modified codons with respect to a natural gene sequenceor a cDNA of the 19 kilodalton C-terminal fragment of a Plasmodiumfalciparum merozoite surface protein 1 (MSP-1) while retaining a naturalamino acid sequence thereof.
 61. A synthetic polynucleotide comprising asynthetic sequence encoding a 19 kilodalton C-terminal fragment of aPlasmodium falciparum merozoite surface protein 1 (MSP-1) having a totalGC content of 40% to 60% consisting essentially of SEQ ID NO:1; and apolynucleotide encoding a signal sequence of a Plasmodium MSP-1 protein.62. The synthetic polynucleotide of claim 61, wherein the syntheticpolynucleotide sequence has a GC content of 50%.
 63. The syntheticpolynucleotide of claim 61, wherein the polynucleotide encoding thesignal sequence is a polynucleotide encoding a Plasmodium falciparumMSP-1 protein.
 64. A synthetic polynucleotide comprising (a) a syntheticsequence encoding a 19 kilodalton C-terminal fragment of a Plasmodiumfalciparum merozoite surface protein 1 (MSP-1) having a total GC contentof 40% to 60% consisting essentially of SEQ ID NO:1; (b) apolynucleotide sequence coding for at most 50 C-terminal amino acids ofa 33 kilodalton N-terminal fragment of a Plasmodium falciparum merozoitesurface protein 1 (MSP-1) positioned upstream of the synthetic sequence;and (c) a polynucleotide encoding a signal sequence of a PlasmodiumMSP-1 protein.
 65. The synthetic polynucleotide of claim 64, wherein thepolynucleotide coding for said C-terminal amino acids has at most 35amino acids.
 66. The synthetic polynucleotide of claim 64, wherein thepolynucleotide coding for said C-terminal amino acids has at most 10amino acids.